ABSTRACT/SUMMARY: Urinary tract infection (UTI): i) is caused by uropathogenic Escherichia coli (UPEC) in over 80% of uncomplicated cases in the United States; ii) primarily affects otherwise healthy females (the lifetime prevalence of UTI in women is 50%); iii) is associated with significant morbidity and economic impact; iv) can become chronically recurrent (20-30% of women diagnosed with a UTI will experience a recurrent UTI (rUTI) in the following months, with some suffering six or more per year). Over 1 million women in the US are referred to urologists each year because of rUTIs and treatment difficulties, which are rising due to the rapid spread of antibiotic resistance in UPEC. Further, 60% of rUTIs are due to the same strain of E. coli that caused the initial infection, arguing that there exist host-associated reservoirs that are recalcitrant to antibiotic treatment and can seed rUTIs. The gastrointestinal tract (GIT) is an important reservoir for E. coli in humans. At the time of UTI, the causal E. coli strain is often the predominant E. coli strain in the GIT, which can persist there even after antibiotic therapy. The healthy GIT microbiota (the collection of microorganisms in the GIT) is a key mediator of homeostasis with the host immune system and can prevent colonization by bacterial pathogens. Ironically, antibiotic treatments meant to clear pathogens can also disrupt the GIT microbiota and expose individuals to an increased risk of colonization by pathogens. This proposal seeks to transform UTI research by investigating the unexplored gut-bladder axis. Goals include elucidating the interplay between UPEC, the GIT microbiota, UPEC pathogenesis and rUTI susceptibility, much of which was previously not technologically feasible. High- resolution longitudinal analyses of the GIT microbiota from rUTI patients have revealed: i) striking differential patterns of UPEC colonization, persistence, and displacement in the GIT; and ii) differences in the GIT microbiota structure of rUTI patients and healthy controls. Further, rUTI patients had an elevated inflammation status, even at baseline. These data have led to the hypothesis that the altered GIT microbiota of women with frequent rUTI may be conducive for UPEC persistence and blooming in the GIT, predisposing to the seeding of UPEC into the bladder to cause rUTIs. We will study the impact of UPEC reservoirs in the GIT and an altered microbiota on mucosal and systemic immunologic changes and the susceptibility and/or host response to rUTIs. This proposal will use clinical samples from rUTI sufferers and healthy controls, newly developed genomic and transcriptomic technologies, and conventional and humanized gnotobiotic mouse models to: Aim 1) unveil factors critical for UPEC colonization of the GIT and the establishment of a reservoir capable of seeding rUTI; Aim 2) elucidate the effects of dysbiotic GIT microbiota found in rUTI patients on host immune functions and UTI susceptibility; and Aim 3) determine the role of the microbiota in controlling UPEC colonization of the GIT These studies will transform the development of antibiotic-sparing therapeutics urgently needed to treat and prevent rUTI.